WO2023072170A1 - A METHOD FOR PROMOTING THE DIFFERENTIATION OF STEM CELLS INTO FUNCTIONALLY MATURE ISLET β CELLS AND USE THEREOF - Google Patents

A METHOD FOR PROMOTING THE DIFFERENTIATION OF STEM CELLS INTO FUNCTIONALLY MATURE ISLET β CELLS AND USE THEREOF Download PDF

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WO2023072170A1
WO2023072170A1 PCT/CN2022/127805 CN2022127805W WO2023072170A1 WO 2023072170 A1 WO2023072170 A1 WO 2023072170A1 CN 2022127805 W CN2022127805 W CN 2022127805W WO 2023072170 A1 WO2023072170 A1 WO 2023072170A1
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cells
islet
medium
differentiation
cell
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PCT/CN2022/127805
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French (fr)
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Yizhe SONG
Jingqiu LI
Zhensheng OU
Leilei GUO
Tingting TANG
Peiwen XING
Jing Liu
Huifang ZHAO
Shidu ZHANG
Qunrui YE
Xiaofeng Chen
Wenjia LI
Shanshan FENG
Jing Li
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Sunshine Lake Pharma Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans

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  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for promoting the differentiation of stem cells into functionally mature islet ⁇ cells and use thereof.
  • Stem cell-derived islet ⁇ cells can provide a useful drug discovery platform for diabetes, and it is also expected to be a cell replacement therapy for diabetes in the future. Therefore, the directed differentiation of human embryonic stem cells or human induced pluripotent stem cells into islet ⁇ cells has attracted widespread attention, and there have been many reports. Many methods were reported for inducing the differentiation of stem cells into islet ⁇ cells, but the differentiation efficiency is generally low, and the resulting islet ⁇ cells are less numerous or functionally immature, which has become a bottleneck in the research of stem cell-derived islet ⁇ cells.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention develops a method for promoting the differentiation of human pluripotent stem cells or human induced pluripotent stem cells into functionally mature islet ⁇ cells based on the differentiation method of stem cells developed by Melton DA and others (Pagliuca, F. W. et al. Generation of functional human pancreatic ⁇ cells in vitro. Cell 159, 428–439 (2014) ) into islet ⁇ cells.
  • the method comprises the addition of M3 muscarinic acetylcholine receptor agonists such as acetylcholine at a specific stage of differentiation of endocrine progenitor cells into islet ⁇ cells, thereby increasing the number of islet ⁇ cells expressing NKX6.1 and C-peptide. That is to say, the addition of M3 muscarinic acetylcholine receptor agonists at the specific stage of differentiation of endocrine progenitor cells into islet ⁇ cells can significantly promote the differentiation of stem cells into functionally mature islet ⁇ cells.
  • M3 muscarinic acetylcholine receptor agonists such as acetylcholine
  • the present invention provides a method for obtaining islet ⁇ cells in vitro.
  • the method comprises: coculturing the endocrine progenitor cells with medium containing an M3 muscarinic acetylcholine receptor agonist, so as to differentiate the endocrine progenitor cells into islet ⁇ cells, wherein the coculture does not occur in the differentiation process from primitive gut tube cells to endocrine progenitor cells.
  • M3 muscarinic acetylcholine receptor agonist at the specific stage of differentiation of endocrine progenitor cells into islet ⁇ cells is different from the prior art -the addition of M3 muscarinic acetylcholine receptor agonists during the differentiation of primitive gut tube cells to islet ⁇ cells and can significantly increase the proportion of NKX6.1 and C-peptide double positive islet ⁇ cells , representing functionally mature islet ⁇ cells.
  • the addition of M3 muscarinic acetylcholine receptor agonist at the specific stage of differentiation of endocrine progenitor cells into islet ⁇ cells can significantly increase the proportion of functionally mature islet ⁇ cells obtained and the differentiate efficiency of stem cells into functionally mature islet ⁇ cells.
  • the endocrine progenitor cells express C-peptide, NKX6.1 and other markers.
  • the above method may further comprise at least one of the following additional technical features:
  • the endocrine progenitor cells are obtained by differentiation of primitive gut tube cells, and in the differentiation process from primitive gut tube cells to endocrine progenitor cells, the cells are not cocultured with medium containing the M3 muscarinic acetylcholine receptor agonist.
  • the M3 muscarinic acetylcholine receptor agonist is acetylcholine.
  • coculturing the endocrine progenitor cells with medium containing the M3 muscarinic acetylcholine receptor agonist is accomplished as follows: the endocrine progenitor cells are differentiated and cultured in S6 complete medium, and the S6 complete medium is MCDB131 medium supplemented with 25-200 ⁇ M acetylcholine.
  • S6 complete medium is MCDB131 medium supplemented with 25-200 ⁇ M acetylcholine.
  • the inventors found that the concentration of acetylcholine in the medium is 25-200 ⁇ M, which can increase the percentage of mature ⁇ cells obtained.
  • the S6 complete medium is MCDB131 medium supplemented with 25-100 ⁇ M acetylcholine.
  • concentration of acetylcholine in the S6 complete medium is 25-100 ⁇ M, which can further significantly increase the percentage of mature ⁇ cells obtained.
  • the S6 complete medium further comprises: 8 mM D- (+) -glucose, 1.23 g/L NaHCO 3 , 2% (mass volume fraction, 2 g/100 mL) BSA, ITS-X at a dilution ratio of 1: 200, 2 mM Glutamax, 0.25 mM vitamin C, and 1% (volume fraction) penicillin and streptomycin (Pen/Strep) .
  • the endocrine progenitor cells are cultured in S6 complete medium for 7 days.
  • the endocrine progenitor cells are obtained as follows: culture the primitive gut tube cells in S3 complete medium for 2 days to obtain PDX1 positive pancreatic progenitor cells; subsequently culture the PDX1 positive pancreatic progenitor cells in S4 complete medium for 5 days to obtain PDX1/NKX6.1 double positive pancreatic progenitor cells; the PDX1/NKX6.1 double positive pancreatic progenitor cells are cultured in S5 complete medium for 7 days to obtain the endocrine progenitor cells.
  • the S3 complete medium is MCDB131 medium containing 8 mM D- (+) -glucose, 1.23 g/L NaHCO 3 , 2%BSA, ITS-X with a dilution ratio of 1: 200, 2 mM Glutamax, 0.25 mM Vitamin C, and 1%Pen/Strep.
  • S4 complete medium supplemented with 50 ng/mL KGF, 0.25 ⁇ M SANT1, 10 ⁇ M Y27632, 5 ng/mL Activin A, and 0.1 ⁇ M RA is used.
  • the medium is completely changed on the first day, the medium is not changed on the second day, the medium is completely changed on the third day, the medium is not changed on the fourth day, and the medium is completely changed on the fifth day.
  • the S5 complete medium is MCDB131 medium containing 20 mM D- (+) -glucose, 1.754 g/L NaHCO 3 , 2%BSA, 0.5%ITS-X, 2 mM Glutamax, 0.25 mM vitamin C, 1%Pen/Strep and 10 ⁇ g/mL heparin.
  • the PDX1/NKX6.1 double positive pancreatic progenitor cells are cultured in the S5 complete medium. On the first day the medium is completely changed, and the S4 complete medium is replaced with the S5 medium.
  • the S5 medium is supplemented with 0.25 ⁇ M SANT1, 0.1 ⁇ M RA, 1 ⁇ M XXI, 10 ⁇ M Alk5i, 1 ⁇ M T3, and 20 ng/mL Betacellulin.
  • the medium is not changed on the second day, the medium containing the same supplemented factors as the first day is continuously used on the third day, and the medium is not changed on the fourth day.
  • the medium is changed to S5 complete medium supplemented with 25 nM RA, 1 ⁇ M XXI, 10 ⁇ M Alk5i, 1 ⁇ M T3 and 20 ng/mL Betacellulin.
  • the medium is not changed on the sixth day, and the medium is completely changed on the seventh day, and it is still replaced with S5 complete medium supplemented with 25 nM RA, 1 ⁇ M XXI, 10 ⁇ M Alk5i, 1 ⁇ M T3, and 20 ng/mL Betacellulin.
  • the primitive gut tube cells are obtained as follows: the starting stem cells are inoculated into mTeSR1 medium, the mTeSR1 medium contains 10 ⁇ M Y27632, wherein the inoculation density of the starting stem cells in a six-well plate is 0.5*10 ⁇ 6 cells/well; the inoculated starting stem cells are cultured for 2 days at 37°C and 5%CO 2 , preferably, the culture process adopts a half medium change method; the subsequent stage 1 and 2 differentiation are performed, adopting half medium change method, so as to obtain the primitive gut tube cells, wherein the stage 1 differentiation lasts for 3 days in S1 complete medium, and the stage 2 differentiation lasts for 3 days in S2 complete medium.
  • the inoculation density of the starting stem cells in the six-well plate is 0.5*10 ⁇ 6 cells/well, which can further improve the differentiation efficiency; adopting the half medium change method instead of the full medium change method during the culture process will further improve the differentiation efficiency of subsequent cells.
  • Endocrine progenitor cells of the invention can be interchanged with the “pancreatic endocrine progenitor cells” , and refer to the pancreatic endodermal cells that can become pancreatic hormone expressing cells. Endocrine progenitor cells can be characterized by their NKX6.1 and C-peptide expression.
  • primordial gut tube cells in the present invention refer to the cells that differentiate from the endoderm and can be differentiated into ⁇ cells (e.g., pancreatic ⁇ cells) .
  • Primitive gut tube cells express at least one of the following markers: FoxA2 or HNF4- ⁇ .
  • Primitive gut tube cells can differentiate into cells including lung, liver, pancreas, stomach and intestine cells.
  • PDX1 positive pancreatic progenitor cells refer to the cells, as pancreatic endoderm (PE) cells, capable of differentiating into SC- ⁇ cells such as pancreatic ⁇ cells.
  • PDX1 positive pancreatic progenitor cells express the marker PDX1.
  • PDX1/NKX6.1 double positive pancreatic progenitor cells refer to the cells, as pancreatic endoderm (PE) cells, capable of differentiating into insulin-producing cells such as pancreatic ⁇ cells.
  • PE pancreatic endoderm
  • PDX1 positive and NKX6.1 positive pancreatic progenitor cells express markers PDX1 and NKX6.1.
  • markers described in the invention can be evaluated by any method known to those skilled in the art, such as immunochemistry, using antibodies or quantitative RT-PCR.
  • half medium change method means that each time the culture medium is changed, only half volume of the original culture medium is changed. For example, if the medium volume is 5 mL, 2.5 mL old culture medium is removed, and 2.5 mL fresh culture medium is added.
  • the “full medium change method” described in this application means that each time the culture medium is changed, all culture medium is removed before adding the fresh medium. For example, if the medium volume is 5 mL, all the old culture medium, and 5 mL fresh medium is added.
  • the starting stem cells are commercial stem cell lines.
  • the commercial stem cell line is a H9 cell line.
  • the starting stem cells are pre-expanded.
  • the amplification process is performed as follows: H9 cell clusters are cultured in suspension using mTeSR1 medium; the H9 cell clusters after suspension culture were digested to obtain H9 single cells; the H9 single cells are inoculated into mTeSR1 medium at a density of 6*10 ⁇ 5 cells/mL and cultured, preferably, the half medium change method is adopted in the culture process; optionally, the cultured cells are subjected to karyotype identification, flow cytometry detection and mycoplasma contamination detection; wherein, the karyotype identification result is normal (that is, the number of chromosomes is 46, and there is no chromosome crossover, exchange, translocation, etc.
  • the flow cytometry detection results show that the proportions of OCT4 and SSEA4 positive cells were not less than 95% (it proves that the stemness of stem cells is well maintained) , and there is no mycoplasma contamination, indicating that the cells after culture are starting stem cells for differentiation.
  • the present invention provides an islet ⁇ cell.
  • the islet ⁇ cell is obtained by the aforementioned method.
  • the proportion of functionally mature islet ⁇ cells is high.
  • the islet ⁇ cell according to the embodiments of the present invention can represent a useful drug discovery platform for the treatment of diabetes, and it is also expected to be developed as a cell therapy preparation for the treatment of diabetes in the future.
  • the present invention provides an islet ⁇ cell cluster.
  • the ratio of NKX6.1/C peptide double positive cell is 20%to 50%, preferably, the ratio of NKX6.1/C peptide double positive cell is 20%to 40%.
  • the ratio of NKX6.1/C peptide double positive cell is high, and the proportion of functionally mature islet ⁇ cells is high.
  • the islet ⁇ cells with the NKX6.1/C-peptide double positive can be obtained in various ways.
  • the islet ⁇ cells can be obtained by using the method described earlier in this application.
  • the islet ⁇ cell cluster according to the embodiments of the present invention can provide a useful drug discovery platform for the treatment of diabetes, and it is also expected to be developed as a cell therapy preparation for the treatment of diabetes in the future.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned islet ⁇ cell or the islet ⁇ cell cluster.
  • the islet ⁇ cells or islet ⁇ cell cluster described could provide a useful drug discovery platform for the treatment of diabetes and are expected to be developed as cell therapy agents for the treatment of diabetes in the future.
  • the pharmaceutical composition includes combinations that are separated in time and/or space, insofar as they can act together to achieve the purpose of the invention.
  • the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately.
  • each component may be administered simultaneously or sequentially to the subject.
  • the “subject” in the present invention generally means mammals, such as primates and/or rodents, especially humans, monkeys or mice.
  • the present invention provides the use of the aforementioned islet ⁇ cell, the islet ⁇ cell cluster or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating or preventing diabetes.
  • the present invention provides a method of treating or preventing diabetes in a subject comprising administering to the subject a therapeutically effective amount of the aforementioned islet ⁇ cell, the islet ⁇ cell cluster, or the aforementioned pharmaceutical composition.
  • the present invention provides the aforementioned islet ⁇ cell, the islet ⁇ cell cluster, or the aforementioned pharmaceutical composition for use in treating or preventing diabetes.
  • Figure 1 is a graph of the karyotyping result according to step 1) of Example 1 of the present invention.
  • Figure 2 is a graph of the proportion of OCT4 and SSEA4 positive cells according to step 1) of Example 1 of the present invention
  • Figure 3 is a graph of the mycoplasma detection result according to step 1) of Example 1 of the present invention.
  • Figure 4 is a graph of the ratio of NKX6.1/C-peptide double positive detected by three repeated flow cytometry in the group without acetylcholine according to Example 1 of the present invention
  • Figure 5 is a graph of the ratio of NKX6.1/C-peptide double positive detected by three repeated flow cytometry in the group with acetylcholine according to Example 1 of the present invention
  • Figure 6 is a comparison of the ratio of NKX6.1/C-peptide double positive in the group with and without acetylcholine according to Example 1 of the present invention.
  • Figure 7 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group without acetylcholine according to Example 2 of the present invention.
  • Figure 8 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 50 ⁇ M acetylcholine according to Example 2 of the present invention.
  • Figure 9 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 100 ⁇ M acetylcholine according to Example 2 of the present invention.
  • Figure 10 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 10 ⁇ M acetylcholine according to Example 3 of the present invention.
  • Figure 11 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 25 ⁇ M acetylcholine according to Example 4 of the present invention.
  • Figure 12 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 10 ⁇ M acetylcholine according to Example 5 of the present invention.
  • Figure 13 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 250 ⁇ M acetylcholine according to Example 6 of the present invention.
  • expression in the attached figures means the expression, “unstained” means the unstained series, “Isotype” means the isotype control series, and “Stained” means the stained series, “control” means the group without acetylcholine.
  • the present invention develops a method for promoting the differentiation of human pluripotent stem cells or human induced pluripotent stem cells into functionally mature islet ⁇ cells based on the differentiation method developed by Melton DA and others of stem cells into islet ⁇ cells, that is, M3 muscarinic acetylcholine receptor agonists such as acetylcholine are added at a specific stage of differentiation of endocrine progenitor cells into islet ⁇ cells, thereby increasing the proportion of islet ⁇ cells expressing NKX6.1 and C-peptide.
  • the invention discloses a method in which an M3 muscarinic acetylcholine receptor agonist acts on the G protein-coupled receptor signaling pathway in islet ⁇ cells, thereby improving the differentiation of stem cells into functionally mature islet ⁇ cells.
  • M3 muscarinic acetylcholine receptor agonists act on the G protein-coupled receptors of islet ⁇ cells, and the signal is transduced to PLC ⁇ , which further activates PKC, Erk1/2, enhances the activity of IRS2, and finally increases the expression of key functional genes in islet ⁇ cells.
  • the efficiency of differentiation of stem cells into functionally mature islet ⁇ cells can be significantly improved; the cost is low and the operation is simple.
  • the differentiation method of islet ⁇ cells used in the following examples is as follows:
  • the starting stem cells were H9 cell line, and the differentiation system was an orbital shaker low-adsorption six-well plate.
  • mTeSR1 medium was used to culture H9 cell clusters in suspension.
  • the suspension culture was performed in 125-mL spinner flasks, with the rotate speed of 70 rpm, volume of 75 mL, under the culture condition of 37 °C and 5%CO 2 .
  • the cells were passaged every 4 days.
  • the H9 cell clusters was digested into single cells uing Accutase, counted, and inoculated into mTeSR1 medium containing 10 ⁇ M Y27632 at cell density of 6*10 ⁇ 5 cells/mL.
  • Half medium change was performed every day during the culture process, and 37.5 mL fresh mTeSR1 medium was added.
  • the H9 cell clusters was digested into single cells with Accutase, counted, and inoculated to a six-well low-adsorption plate at density of 0.5*10 ⁇ 6 cells/well using mTeSR1 medium containing 10 ⁇ M Y27632 (5.5 ml per well) , with a speed of 100 rpm, and cultured under the culture condition of 37 °C and 5%CO 2 .
  • Stage 0 was for two days. The medium was changed every day. The half medium change method was adopted, 3 mL of old medium was removed, and 3 mL of fresh mTeSR1 medium was added;
  • Stage 1 (S1) medium MCDB131 + 8 mM D- (+) -glucose + 2.46 g/L NaHCO 3 + 2%BSA + 0.002%(1:50000) ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
  • Stage 2 (S2) medium MCDB131 + 8 mM D- (+) -glucose + 1.23 g/L NaHCO 3 + 2%BSA + 0.002%ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
  • Stage 3 (S3) medium MCDB131 + 8 mM D- (+) -glucose + 1.23 g/L NaHCO3 + 2%BSA + 0.5% (1: 200) ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
  • Stage 4 (S4) medium MCDB131+8 mM D- (+) -glucose +1.23 g/L NaHCO 3 + 2%BSA+ 0.5%ITS-X +2 mM Glutamax+0.25 mM vitamin C+ 1%Pen/Strep;
  • Stage 5 (S5) medium MCDB131 + 20 mM D- (+) -glucose + 1.754 g/L NaHCO3 + 2%BSA + 0.5%ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep + 10 ⁇ g/mL heparin.
  • stage 5 of differentiation the cell clusters were digested into single cells using TrypLETM Express digestion solution, and then re-inoculated into a low-adsorption six-well plate with a culture volume of 5.5 mL, and the cell inoculation density was 1*10 ⁇ 6 cells/mL.
  • Stage 6 (S6) medium MCDB131+8 mM D- (+) -glucose+1.23 g/L NaHCO3+2%BSA+ 0.5%ITS-X+2 mM Glutamax+0.25 mM vitamin C+1%Pen/Strep was used, with additional addition of 10-200 ⁇ M acetylcholine as S6 complete medium.
  • the culture conditions were unchanged, the S6 complete medium was replaced every other day, and the full medium change method was used for 7 days (D1 ⁇ D7) of culture.
  • the differentiated cell clusters were digested into single cells, and the proportion of NKX6.1 and C-peptide double-positive cell populations, i.e., the proportion of terminally differentiated mature insulin-producing cells, was detected by flow cytometry.
  • the digested single cell sample 2*10 ⁇ 6 cells obtained in step 4) were collected, and filtered through a 70 ⁇ m mesh.
  • the cells were washed twice with 1 mL PBS, centrifuged at 200 g for 4 min; fix: 1 mL BD Cytofix fixation buffer was added and the mixture was fixed at room temperature for 20 min; permeabilization: the mixture was centrifuged at 800 g for 4 min to remove the fixative, then washed twice with 1 mL of Perm Wash Buffer (10-fold dilution with water filtered through a 0.22 ⁇ m filter) , centrifuged at 800 g for 4 min, 300 ⁇ L of Perm Wash Buffer was added to permeabilize for 30 min.
  • Unstained cells Isotype control, and Stain cells; staining: Unstained cells group was left untreated, Isotype control group was added with 0.625 ⁇ L PE Mouse IgG1, ⁇ Isotype Control and 5 ⁇ L Alexa Fluor 647 Mouse IgG1 ⁇ Isotype Control, Stain cells group was added with 5 ⁇ L Alexa Fluor 647 Mouse Anti-C-Peptide and 5 ⁇ L PE Mouse Anti-Nkx6.1.
  • NKX6.1 and C-peptide were detected by flow cytometry (BD FASVerse) within 2 hours.
  • the starting H9 stem cells were processed from step 1) to 3) , and the karyotype of the obtained cells, the proportion of OCT4 and SSEA4 positive cells, and mycoplasma were detected during step 1) .
  • the detection results are shown in Figures 1-3. The results showed that the cell karyotype was normal, the proportion of OCT4 and SSEA4 positive cells was above 95%, and there was no mycoplasma contamination.
  • stage 6 i.e., step 4)
  • the cells were divided into two groups: 1) group without acetylcholine; 2) group with 25 ⁇ M acetylcholine; there were three replicate wells for each group.
  • the proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively; the three duplicate wells in the group with acetylcholine were 22.30%, 25.97%and 26.60%, respectively.
  • the specific flow cytometry results are shown in Figures 4-6. After T-test analysis, the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was significantly higher than that in the group without acetylcholine, indicating that the addition of 25 ⁇ M acetylcholine at stage 6 could increase the proportion of stem cells that differentiate into mature insulin-producing cells.
  • the starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) , they were divided into three groups: 1) group without acetylcholine; 2) group with 50 ⁇ M acetylcholine; 3) group with 100 ⁇ M acetylcholine.
  • the proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the group without acetylcholine was 21.79%; the group with 50 ⁇ M acetylcholine was 30.02%; the group with 100 ⁇ M acetylcholine was 39.86%.
  • the specific flow cytometry results are shown in Figures 7-9.
  • the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was significantly higher than that in the group without acetylcholine, and with the increase of the concentration, the double positive ratio increased, indicating that adding 50 ⁇ M or 100 ⁇ M acetylcholine at stage 6 could increase the proportion of stem cells differentiated into insulin-producing cells.
  • the difference between the experimental operation of the group with acetylcholineand the group with acetylcholine in Example 1 was that 10 ⁇ M acetylcholine was added to the cell culture medium at stage 4 (differentiation of PDX1 positive pancreatic progenitor cells into PDX1/NKX6.1 double positive pancreatic progenitor cells) and stage 5 (differentiation of PDX1/NKX6.1 double positive pancreatic progenitor cells into endocrine progenitor cells) respectively.
  • the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was not significantly higher than that in the group without acetylcholine, indicating that the addition of acetylcholine at stage 4 and stage 5 of differentiation had no effect on the differentiation of stem cells into mature insulin-producing cells.
  • the difference between the experimental operation of the group with acetylcholineand the group with acetylcholine in Example 1 was that 25 ⁇ M acetylcholine was added to the cell culture medium at stage 4 (differentiation of PDX1 positive pancreatic progenitor cells into PDX1/NKX6.1 double positive pancreatic progenitor cells) and stage 5 (differentiation of PDX1/NKX6.1 double positive pancreatic progenitor cells into endocrine progenitor cells) respectively.
  • the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was not significantly higher than that in the group without acetylcholine, indicating that the addition of acetylcholine at stage 4 and stage 5 of differentiation had no effect on the differentiation of stem cells into mature insulin-producing cells.
  • the starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) ) , they were divided into two groups: 1) group without acetylcholine; 2) group with 10 ⁇ M acetylcholine; there were three replicate wells for each group.
  • the proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively (see Figure 4) ; the three duplicate wells in the group with 10 ⁇ M acetylcholine were 15.06%, 17.52%and 22.95%, respectively (see Figure 12) .
  • the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was not significantly higher than that in the group without acetylcholine, indicating that the addition of 10 ⁇ M acetylcholine at stage 6 had no effect on the differentiation of stem cells into mature insulin-producing cells.
  • the starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) ) , they were divided into two groups: 1) group without acetylcholine; 2) group with 250 ⁇ M acetylcholine.
  • the proportion of insulin-producing cells was detected by flow cytometry: the group without acetylcholine was 21.79% (see Figure 7) ; the group with 250 ⁇ M acetylcholine was 21.46%on average (see Figure 13) .
  • the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine did not increase compared with the group without acetylcholine, indicating that adding 250 ⁇ M acetylcholine at stage 6 had no effect on the differentiation of stem cells into mature insulin-producing cells.

Abstract

Provided is a method for promoting the differentiation of pluripotent stem cells into functionally mature islet β cells. The method comprises: coculturing endocrine progenitor cells with medium containing an M3 muscarinic acetylcholine receptor agonist, so as to facilitate the differentiation of endocrine progenitor cells into islet β cells; wherein, the endocrine progenitor cells are generated from primitive gut tube cells through in vitro differentiation. During differentiation process from primitive gut tube cells to endocrine progenitor cells, the cells are not cocultured with medium containing the M3 muscarinic acetylcholine receptor agonist.

Description

A METHOD FOR PROMOTING THE DIFFERENTIATION OF STEM CELLS INTO FUNCTIONALLY MATURE ISLET β CELLS AND USE THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the priority and benefit of Chinese Patent Application No. 202111264612.8, filed with the State Intellectual Property Office of China on October 28, 2021, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for promoting the differentiation of stem cells into functionally mature islet β cells and use thereof.
BACKGROUND ART
Stem cell-derived islet β cells can provide a useful drug discovery platform for diabetes, and it is also expected to be a cell replacement therapy for diabetes in the future. Therefore, the directed differentiation of human embryonic stem cells or human induced pluripotent stem cells into islet β cells has attracted widespread attention, and there have been many reports. Many methods were reported for inducing the differentiation of stem cells into islet β cells, but the differentiation efficiency is generally low, and the resulting islet β cells are less numerous or functionally immature, which has become a bottleneck in the research of stem cell-derived islet β cells.
How to improve the differentiation efficiency of stem cells into functionally mature islet β cells is an urgent problem to be solved.
SUMMARY
The present invention aims to solve one of the technical problems in the related art at least to a certain extent. In order to solve the problem of low efficiency of mature islet β cells differentiation from stem cells, the present invention develops a method for promoting the differentiation of human pluripotent stem cells or human induced pluripotent stem cells into functionally mature islet β cells based on the differentiation method of stem cells developed by Melton DA and others (Pagliuca, F. W. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2014) ) into islet β cells. The method comprises the  addition of M3 muscarinic acetylcholine receptor agonists such as acetylcholine at a specific stage of differentiation of endocrine progenitor cells into islet β cells, thereby increasing the number of islet β cells expressing NKX6.1 and C-peptide. That is to say, the addition of M3 muscarinic acetylcholine receptor agonists at the specific stage of differentiation of endocrine progenitor cells into islet β cells can significantly promote the differentiation of stem cells into functionally mature islet β cells.
In the first aspect of the present invention, the present invention provides a method for obtaining islet β cells in vitro. According to an embodiment of the present invention, the method comprises: coculturing the endocrine progenitor cells with medium containing an M3 muscarinic acetylcholine receptor agonist, so as to differentiate the endocrine progenitor cells into islet β cells, wherein the coculture does not occur in the differentiation process from primitive gut tube cells to endocrine progenitor cells. The inventors found that the addition of M3 muscarinic acetylcholine receptor agonist at the specific stage of differentiation of endocrine progenitor cells into islet β cells is different from the prior art -the addition of M3 muscarinic acetylcholine receptor agonists during the differentiation of primitive gut tube cells to islet β cells and can significantly increase the proportion of NKX6.1 and C-peptide double positive islet β cells , representing functionally mature islet β cells. That is to say, the addition of M3 muscarinic acetylcholine receptor agonist at the specific stage of differentiation of endocrine progenitor cells into islet β cells can significantly increase the proportion of functionally mature islet β cells obtained and the differentiate efficiency of stem cells into functionally mature islet β cells. Wherein, the endocrine progenitor cells express C-peptide, NKX6.1 and other markers.
According to an embodiment of the present invention, the above method may further comprise at least one of the following additional technical features:
According to an embodiment of the present invention, the endocrine progenitor cells are obtained by differentiation of primitive gut tube cells, and in the differentiation process from primitive gut tube cells to endocrine progenitor cells, the cells are not cocultured with medium containing the M3 muscarinic acetylcholine receptor agonist.
According to an embodiment of the present invention, the M3 muscarinic acetylcholine receptor agonist is acetylcholine.
According to an embodiment of the present invention, coculturing the endocrine progenitor cells with medium containing the M3 muscarinic acetylcholine receptor agonist is accomplished as follows: the endocrine progenitor cells are differentiated and cultured in S6 complete medium, and the S6 complete medium is MCDB131 medium supplemented with 25-200 μM acetylcholine. The inventors found that the  concentration of acetylcholine in the medium is 25-200 μM, which can increase the percentage of mature βcells obtained.
According to an embodiment of the present invention, the S6 complete medium is MCDB131 medium supplemented with 25-100 μM acetylcholine. The inventors found that the concentration of acetylcholine in the S6 complete medium is 25-100 μM, which can further significantly increase the percentage of mature β cells obtained.
According to an embodiment of the present invention, the S6 complete medium further comprises: 8 mM D- (+) -glucose, 1.23 g/L NaHCO 3, 2% (mass volume fraction, 2 g/100 mL) BSA, ITS-X at a dilution ratio of 1: 200, 2 mM Glutamax, 0.25 mM vitamin C, and 1% (volume fraction) penicillin and streptomycin (Pen/Strep) .
According to an embodiment of the present invention, the endocrine progenitor cells are cultured in S6 complete medium for 7 days. The inventor found that when the endocrine progenitor cells are cultured in S6 complete medium for 7 days, the number of mature islet β cells will further increase.
According to an embodiment of the present invention, the endocrine progenitor cells are obtained as follows: culture the primitive gut tube cells in S3 complete medium for 2 days to obtain PDX1 positive pancreatic progenitor cells; subsequently culture the PDX1 positive pancreatic progenitor cells in S4 complete medium for 5 days to obtain PDX1/NKX6.1 double positive pancreatic progenitor cells; the PDX1/NKX6.1 double positive pancreatic progenitor cells are cultured in S5 complete medium for 7 days to obtain the endocrine progenitor cells.
According to a specific embodiment of the present invention, the S3 complete medium is MCDB131 medium containing 8 mM D- (+) -glucose, 1.23 g/L NaHCO 3, 2%BSA, ITS-X with a dilution ratio of 1: 200, 2 mM Glutamax, 0.25 mM Vitamin C, and 1%Pen/Strep. During the 2-day culture of primitive gut tube cells in S3 complete medium, 200 nM LDN193189, 50 ng/mL KGF, 0.25 μM SANT1, 500 nM PdBU, 10 μM Y27632 and 2 μM RA are added on the first day; on the second day, the medium is completely changed, that is, the medium of the first day is removed and replaced using the same volume of S3 complete medium supplemented with 50 ng/mL KGF, 0.25 μM SANT1, 500 nM PdBU, 10 μM Y27632 and 2 μM RA. The PDX1 positive pancreatic progenitor cells are cultured in S4 complete medium for 5 days. From the first day to the fifth day, S4 complete medium supplemented with 50 ng/mL KGF, 0.25 μM SANT1, 10 μM Y27632, 5 ng/mL Activin A, and 0.1 μM RA is used. The medium is completely changed on the first day, the medium is not changed on the second day, the medium is completely changed on the third day, the medium is not changed on the fourth day, and the medium is completely changed on the fifth day. The S5 complete medium is  MCDB131 medium containing 20 mM D- (+) -glucose, 1.754 g/L NaHCO 3, 2%BSA, 0.5%ITS-X, 2 mM Glutamax, 0.25 mM vitamin C, 1%Pen/Strep and 10 μg/mL heparin. The PDX1/NKX6.1 double positive pancreatic progenitor cells are cultured in the S5 complete medium. On the first day the medium is completely changed, and the S4 complete medium is replaced with the S5 medium. The S5 medium is supplemented with 0.25 μM SANT1, 0.1 μM RA, 1 μM XXI, 10 μM Alk5i, 1 μM T3, and 20 ng/mL Betacellulin. The medium is not changed on the second day, the medium containing the same supplemented factors as the first day is continuously used on the third day, and the medium is not changed on the fourth day. On the fifth day, the medium is changed to S5 complete medium supplemented with 25 nM RA, 1 μM XXI, 10 μM Alk5i, 1 μM T3 and 20 ng/mL Betacellulin. The medium is not changed on the sixth day, and the medium is completely changed on the seventh day, and it is still replaced with S5 complete medium supplemented with 25 nM RA, 1 μM XXI, 10 μM Alk5i, 1 μM T3, and 20 ng/mL Betacellulin.
According to an embodiment of the present invention, the primitive gut tube cells are obtained as follows: the starting stem cells are inoculated into mTeSR1 medium, the mTeSR1 medium contains 10 μM Y27632, wherein the inoculation density of the starting stem cells in a six-well plate is 0.5*10^6 cells/well; the inoculated starting stem cells are cultured for 2 days at 37℃ and 5%CO 2, preferably, the culture process adopts a half medium change method; the  subsequent stage  1 and 2 differentiation are performed, adopting half medium change method, so as to obtain the primitive gut tube cells, wherein the stage 1 differentiation lasts for 3 days in S1 complete medium, and the stage 2 differentiation lasts for 3 days in S2 complete medium. The inventors found that the inoculation density of the starting stem cells in the six-well plate is 0.5*10^6 cells/well, which can further improve the differentiation efficiency; adopting the half medium change method instead of the full medium change method during the culture process will further improve the differentiation efficiency of subsequent cells.
It should be noted that the “endocrine progenitor cells” of the invention can be interchanged with the “pancreatic endocrine progenitor cells” , and refer to the pancreatic endodermal cells that can become pancreatic hormone expressing cells. Endocrine progenitor cells can be characterized by their NKX6.1 and C-peptide expression.
It should be noted that the “primitive gut tube cells” in the present invention refer to the cells that differentiate from the endoderm and can be differentiated into β cells (e.g., pancreatic β cells) . Primitive gut tube cells express at least one of the following markers: FoxA2 or HNF4-α. Primitive gut tube cells can differentiate into cells including lung, liver, pancreas, stomach and intestine cells.
It should be noted that the “PDX1 positive pancreatic progenitor cells” mentioned in the  invention refer to the cells, as pancreatic endoderm (PE) cells, capable of differentiating into SC-β cells such as pancreatic β cells. PDX1 positive pancreatic progenitor cells express the marker PDX1.
It should be noted that the “PDX1/NKX6.1 double positive pancreatic progenitor cells” mentioned in the invention refer to the cells, as pancreatic endoderm (PE) cells, capable of differentiating into insulin-producing cells such as pancreatic β cells. PDX1 positive and NKX6.1 positive pancreatic progenitor cells express markers PDX1 and NKX6.1.
It should be noted that the markers described in the invention can be evaluated by any method known to those skilled in the art, such as immunochemistry, using antibodies or quantitative RT-PCR.
It should be noted that the “half medium change method” described in this application means that each time the culture medium is changed, only half volume of the original culture medium is changed. For example, if the medium volume is 5 mL, 2.5 mL old culture medium is removed, and 2.5 mL fresh culture medium is added.
It should be noted that the “full medium change method” described in this application means that each time the culture medium is changed, all culture medium is removed before adding the fresh medium. For example, if the medium volume is 5 mL, all the old culture medium, and 5 mL fresh medium is added.
According to an embodiment of the present invention, the starting stem cells are commercial stem cell lines. According to a specific embodiment of the present invention, the commercial stem cell line is a H9 cell line.
According to an embodiment of the present invention, the starting stem cells are pre-expanded.
According to a specific embodiment of the present invention, the amplification process is performed as follows: H9 cell clusters are cultured in suspension using mTeSR1 medium; the H9 cell clusters after suspension culture were digested to obtain H9 single cells; the H9 single cells are inoculated into mTeSR1 medium at a density of 6*10^5 cells/mL and cultured, preferably, the half medium change method is adopted in the culture process; optionally, the cultured cells are subjected to karyotype identification, flow cytometry detection and mycoplasma contamination detection; wherein, the karyotype identification result is normal (that is, the number of chromosomes is 46, and there is no chromosome crossover, exchange, translocation, etc. ) , the flow cytometry detection results show that the proportions of OCT4 and SSEA4 positive cells were not less than 95% (it proves that the stemness of stem cells is well maintained) , and there is no mycoplasma contamination, indicating that the cells after culture are starting stem cells for differentiation.
In the second aspect of the present invention, the present invention provides an islet β cell. According to an embodiment of the present invention, the islet β cell is obtained by the aforementioned  method. According to the islet β cells of the embodiments of the present invention, the proportion of functionally mature islet β cells is high. The islet β cell according to the embodiments of the present invention can represent a useful drug discovery platform for the treatment of diabetes, and it is also expected to be developed as a cell therapy preparation for the treatment of diabetes in the future.
In the third aspect of the present invention, the present invention provides an islet β cell cluster. According to an embodiment of the present invention, in the cell cluster, the ratio of NKX6.1/C peptide double positive cell is 20%to 50%, preferably, the ratio of NKX6.1/C peptide double positive cell is 20%to 40%. According to the islet β cell cluster of the embodiment of the present invention, the ratio of NKX6.1/C peptide double positive cell is high, and the proportion of functionally mature islet β cells is high. Wherein, the islet βcells with the NKX6.1/C-peptide double positive can be obtained in various ways. For example, the islet βcells can be obtained by using the method described earlier in this application. The islet β cell cluster according to the embodiments of the present invention can provide a useful drug discovery platform for the treatment of diabetes, and it is also expected to be developed as a cell therapy preparation for the treatment of diabetes in the future.
In the fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned islet β cell or the islet β cell cluster. As previously mentioned, the islet β cells or islet β cell cluster described could provide a useful drug discovery platform for the treatment of diabetes and are expected to be developed as cell therapy agents for the treatment of diabetes in the future.
The pharmaceutical composition includes combinations that are separated in time and/or space, insofar as they can act together to achieve the purpose of the invention. For example, the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately. When the components contained in the pharmaceutical composition are administered separately to the subject, each component may be administered simultaneously or sequentially to the subject.
The “subject” in the present invention generally means mammals, such as primates and/or rodents, especially humans, monkeys or mice.
In the fifth aspect of the present invention, the present invention provides the use of the aforementioned islet β cell, the islet β cell cluster or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating or preventing diabetes.
In the fifth aspect of the present invention, the present invention provides a method of treating or preventing diabetes in a subject comprising administering to the subject a therapeutically effective amount  of the aforementioned islet β cell, the islet β cell cluster, or the aforementioned pharmaceutical composition.
In the fifth aspect of the present invention, the present invention provides the aforementioned islet β cell, the islet β cell cluster, or the aforementioned pharmaceutical composition for use in treating or preventing diabetes.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of the karyotyping result according to step 1) of Example 1 of the present invention;
Figure 2 is a graph of the proportion of OCT4 and SSEA4 positive cells according to step 1) of Example 1 of the present invention;
Figure 3 is a graph of the mycoplasma detection result according to step 1) of Example 1 of the present invention;
Figure 4 is a graph of the ratio of NKX6.1/C-peptide double positive detected by three repeated flow cytometry in the group without acetylcholine according to Example 1 of the present invention;
Figure 5 is a graph of the ratio of NKX6.1/C-peptide double positive detected by three repeated flow cytometry in the group with acetylcholine according to Example 1 of the present invention;
Figure 6 is a comparison of the ratio of NKX6.1/C-peptide double positive in the group with and without acetylcholine according to Example 1 of the present invention;
Figure 7 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group without acetylcholine according to Example 2 of the present invention;
Figure 8 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 50 μM acetylcholine according to Example 2 of the present invention;
Figure 9 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 100 μM acetylcholine according to Example 2 of the present invention;
Figure 10 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 10 μM acetylcholine according to Example 3 of the present invention;
Figure 11 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 25 μM acetylcholine according to Example 4 of the present invention;
Figure 12 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow cytometry in the group with 10 μM acetylcholine according to Example 5 of the present invention; and
Figure 13 is a graph of the ratio of NKX6.1/C-peptide double positive detected by flow  cytometry in the group with 250 μM acetylcholine according to Example 6 of the present invention.
“expression” in the attached figures means the expression, “unstained” means the unstained series, “Isotype” means the isotype control series, and “Stained” means the stained series, “control” means the group without acetylcholine.
EXAMPLES
The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention.
In order to solve the problem of low differentiation efficiency of stem cells into functionally mature islet β cells, the present invention develops a method for promoting the differentiation of human pluripotent stem cells or human induced pluripotent stem cells into functionally mature islet β cells based on the differentiation method developed by Melton DA and others of stem cells into islet β cells, that is, M3 muscarinic acetylcholine receptor agonists such as acetylcholine are added at a specific stage of differentiation of endocrine progenitor cells into islet β cells, thereby increasing the proportion of islet β cells expressing NKX6.1 and C-peptide.
The invention discloses a method in which an M3 muscarinic acetylcholine receptor agonist acts on the G protein-coupled receptor signaling pathway in islet β cells, thereby improving the differentiation of stem cells into functionally mature islet β cells. At the specific stage of differentiation of endocrine progenitor cells into islet β cells, M3 muscarinic acetylcholine receptor agonists act on the G protein-coupled receptors of islet β cells, and the signal is transduced to PLCβ, which further activates PKC, Erk1/2, enhances the activity of IRS2, and finally increases the expression of key functional genes in islet β cells.
By adding the M3 muscarinic acetylcholine receptor agonist at a specific stage, the efficiency of differentiation of stem cells into functionally mature islet β cells can be significantly improved; the cost is low and the operation is simple.
The technical solutions of the present invention will be described in detail below with reference to specific embodiments, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention. Where no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the  field or in accordance with the product instructions shall be carried out. The reagents or instruments used are conventional products that can be purchased from the market if the manufacturer is not indicated.
The differentiation method of islet β cells used in the following examples is as follows:
The starting stem cells were H9 cell line, and the differentiation system was an orbital shaker low-adsorption six-well plate.
1) Cell culture: mTeSR1 medium was used to culture H9 cell clusters in suspension. The suspension culture was performed in 125-mL spinner flasks, with the rotate speed of 70 rpm, volume of 75 mL, under the culture condition of 37 ℃ and 5%CO 2. The cells were passaged every 4 days. During passaging the H9 cell clusters was digested into single cells uing Accutase, counted, and inoculated into mTeSR1 medium containing 10 μM Y27632 at cell density of 6*10^5 cells/mL. Half medium change was performed every day during the culture process, and 37.5 mL fresh mTeSR1 medium was added. Karyotype, stemness and sterity of cells were verified by karyotyping, flow cytometry detection and mycoplasma contamination detection. After all indicators were normal (karyotype identification results were 46, XX (female) , the proportions of OCT4 and SSEA4 positive cells were above 95%, and there was no mycoplasma contamination) , the following steps were performed;
2) At stage 0 before differentiation, the H9 cell clusters was digested into single cells with Accutase, counted, and inoculated to a six-well low-adsorption plate at density of 0.5*10^6 cells/well using mTeSR1 medium containing 10 μM Y27632 (5.5 ml per well) , with a speed of 100 rpm, and cultured under the culture condition of 37 ℃ and 5%CO 2. Stage 0 was for two days. The medium was changed every day. The half medium change method was adopted, 3 mL of old medium was removed, and 3 mL of fresh mTeSR1 medium was added;
3) In order to differentiate the stem cells into insulin-producing cells, the subsequent use of the differentiation medium was carried out in full medium change method, that is, 5.5 mL of the old medium was removed, and 5.5 mL of the corresponding differentiation medium was added. The medium preparation and added differentiation factor of the specific differentiation stages (Stage 1 to Stage 5) are as follows and shown in Table 1:
Stage 1 (S1) medium: MCDB131 + 8 mM D- (+) -glucose + 2.46 g/L NaHCO 3 + 2%BSA + 0.002%(1:50000) ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
Stage 2 (S2) medium: MCDB131 + 8 mM D- (+) -glucose + 1.23 g/L NaHCO 3 + 2%BSA + 0.002%ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
Stage 3 (S3) medium: MCDB131 + 8 mM D- (+) -glucose + 1.23 g/L NaHCO3 + 2%BSA + 0.5% (1: 200)  ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep;
Stage 4 (S4) medium: MCDB131+8 mM D- (+) -glucose +1.23 g/L NaHCO 3+ 2%BSA+ 0.5%ITS-X +2 mM Glutamax+0.25 mM vitamin C+ 1%Pen/Strep;
Stage 5 (S5) medium: MCDB131 + 20 mM D- (+) -glucose + 1.754 g/L NaHCO3 + 2%BSA + 0.5%ITS-X + 2 mM Glutamax + 0.25 mM vitamin C + 1%Pen/Strep + 10 μg/mL heparin.
Table 1
Figure PCTCN2022127805-appb-000001
Figure PCTCN2022127805-appb-000002
Note: No Feed means no medium change. PdBu: phorbol 12, 13-dibutyrate; RA: Retinoic acid; XXI: γ-secretase inhibitor XXI; AlK5i: AlK5 inhibitor (RepSox) ; T3: L-3, 3′, 5-triiodothyronine sodium salt.
4) At the end of stage 5 of differentiation, the cell clusters were digested into single cells using TrypLETM Express digestion solution, and then re-inoculated into a low-adsorption six-well plate with a culture volume of 5.5 mL, and the cell inoculation density was 1*10^6 cells/mL. Stage 6 (S6) medium:  MCDB131+8 mM D- (+) -glucose+1.23 g/L NaHCO3+2%BSA+ 0.5%ITS-X+2 mM Glutamax+0.25 mM vitamin C+1%Pen/Strep was used, with additional addition of 10-200 μM acetylcholine as S6 complete medium. The culture conditions were unchanged, the S6 complete medium was replaced every other day, and the full medium change method was used for 7 days (D1～D7) of culture. The differentiated cell clusters were digested into single cells, and the proportion of NKX6.1 and C-peptide double-positive cell populations, i.e., the proportion of terminally differentiated mature insulin-producing cells, was detected by flow cytometry.
5) The digested single cell sample 2*10^6 cells obtained in step 4) were collected, and filtered through a 70 μm mesh. The cells were washed twice with 1 mL PBS, centrifuged at 200 g for 4 min; fix: 1 mL BD Cytofix fixation buffer was added and the mixture was fixed at room temperature for 20 min; permeabilization: the mixture was centrifuged at 800 g for 4 min to remove the fixative, then washed twice with 1 mL of Perm Wash Buffer (10-fold dilution with water filtered through a 0.22 μm filter) , centrifuged at 800 g for 4 min, 300 μL of Perm Wash Buffer was added to permeabilize for 30 min. The mixture was divided into three groups on average: Unstained cells, Isotype control, and Stain cells; staining: Unstained cells group was left untreated, Isotype control group was added with 0.625 μL PE Mouse IgG1, κ Isotype Control and 5 μL Alexa Fluor 647 Mouse IgG1 κ Isotype Control, Stain cells group was added with 5 μL Alexa Fluor 647 Mouse Anti-C-Peptide and 5 μL PE Mouse Anti-Nkx6.1. After mixing, they were incubated at room temperature for 30 min in the dark, during which the sample was mixed by pipetting every 5 min; washing and resuspending the cells: the mixture was centrifuged at 800 g for 4 min, and the supernatant was removed. The cells were washed twice with 1 mL of Perm Wash Buffer, and centrifuged at 800 g for 4 min. The cells were resuspended in 200 μL BD Pharmingen stain buffer (FBS) and tested on the machine. The expression of NKX6.1 and C-peptide was detected by flow cytometry (BD FASVerse) within 2 hours.
Example 1
The starting H9 stem cells were processed from step 1) to 3) , and the karyotype of the obtained cells, the proportion of OCT4 and SSEA4 positive cells, and mycoplasma were detected during step 1) . The detection results are shown in Figures 1-3. The results showed that the cell karyotype was normal, the proportion of OCT4 and SSEA4 positive cells was above 95%, and there was no mycoplasma contamination. Then at stage 6 (i.e., step 4) ) , the cells were divided into two groups: 1) group without acetylcholine; 2) group with 25 μM acetylcholine; there were three replicate wells for each group. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively; the three duplicate wells in the group with acetylcholine were 22.30%, 25.97%and 26.60%, respectively. The specific flow  cytometry results are shown in Figures 4-6. After T-test analysis, the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was significantly higher than that in the group without acetylcholine, indicating that the addition of 25 μM acetylcholine at stage 6 could increase the proportion of stem cells that differentiate into mature insulin-producing cells.
Example 2
The starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) , they were divided into three groups: 1) group without acetylcholine; 2) group with 50 μM acetylcholine; 3) group with 100 μM acetylcholine. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the group without acetylcholine was 21.79%; the group with 50 μM acetylcholine was 30.02%; the group with 100 μM acetylcholine was 39.86%. The specific flow cytometry results are shown in Figures 7-9. The ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was significantly higher than that in the group without acetylcholine, and with the increase of the concentration, the double positive ratio increased, indicating that adding 50 μM or 100 μM acetylcholine at stage 6 could increase the proportion of stem cells differentiated into insulin-producing cells.
Example 3
The experiments of this example were divided into two groups: 1) group without acetylcholine; 2) group with 10 μM acetylcholine;
The experimental operation of the group without acetylcholine was the same as that of the group without acetylcholine in Example 1;
The difference between the experimental operation of the group with acetylcholineand the group with acetylcholine in Example 1 was that 10 μM acetylcholine was added to the cell culture medium at stage 4 (differentiation of PDX1 positive pancreatic progenitor cells into PDX1/NKX6.1 double positive pancreatic progenitor cells) and stage 5 (differentiation of PDX1/NKX6.1 double positive pancreatic progenitor cells into endocrine progenitor cells) respectively.
There were three replicate wells for each group. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively (see Figure 4) ; the three duplicate wells in the group with 10 μM acetylcholine were 12.99%, 13.46%, and 18.86%, respectively (see Figure 10) . After T-test analysis, the ratio of NKX6.1/C-peptide double positive in the group with  acetylcholine was not significantly higher than that in the group without acetylcholine, indicating that the addition of acetylcholine at stage 4 and stage 5 of differentiation had no effect on the differentiation of stem cells into mature insulin-producing cells.
Example 4
The experiments of this example were divided into two groups: 1) group without acetylcholine; 2) group with 25 μM acetylcholine;
The experimental operation of the group without acetylcholine was the same as that of the group without acetylcholine in Example 1;
The difference between the experimental operation of the group with acetylcholineand the group with acetylcholine in Example 1 was that 25 μM acetylcholine was added to the cell culture medium at stage 4 (differentiation of PDX1 positive pancreatic progenitor cells into PDX1/NKX6.1 double positive pancreatic progenitor cells) and stage 5 (differentiation of PDX1/NKX6.1 double positive pancreatic progenitor cells into endocrine progenitor cells) respectively.
There were three replicate wells for each group. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively (see Figure 4) ; the three duplicate wells in the group with 25 μM acetylcholine were 21.21%, 19.41%, and 16.14%, respectively (see Figure 11) . After T-test analysis, the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was not significantly higher than that in the group without acetylcholine, indicating that the addition of acetylcholine at stage 4 and stage 5 of differentiation had no effect on the differentiation of stem cells into mature insulin-producing cells.
Example 5
The starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) ) , they were divided into two groups: 1) group without acetylcholine; 2) group with 10 μM acetylcholine; there were three replicate wells for each group. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the three replicate wells in the group without acetylcholine were 16.12%, 13.55%and 18.80%, respectively (see Figure 4) ; the three duplicate wells in the group with 10 μM acetylcholine were 15.06%, 17.52%and 22.95%, respectively (see Figure 12) . After T-test analysis, the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine was not significantly  higher than that in the group without acetylcholine, indicating that the addition of 10 μM acetylcholine at stage 6 had no effect on the differentiation of stem cells into mature insulin-producing cells.
Example 6
The starting H9 stem cells were processed from step 1) to 3) , and at stage 6 (i.e., step 4) ) , they were divided into two groups: 1) group without acetylcholine; 2) group with 250 μM acetylcholine. The proportion of insulin-producing cells (the ratio of NKX6.1 and C-peptide double positive) was detected by flow cytometry: the group without acetylcholine was 21.79% (see Figure 7) ; the group with 250 μM acetylcholine was 21.46%on average (see Figure 13) . After analysis, the ratio of NKX6.1/C-peptide double positive in the group with acetylcholine did not increase compared with the group without acetylcholine, indicating that adding 250 μM acetylcholine at stage 6 had no effect on the differentiation of stem cells into mature insulin-producing cells.
Reference throughout this specification to “an embodiment” , “some embodiments” , “one embodiment” , “another example” , “an example” , “a specific example” , or “some examples” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments” , “in one embodiment” , “in an embodiment” , “in another example” , “in an example” , “in a specific example” , or “in some examples” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can integrate and combine different embodiments, examples or the features of them as long as they are not contradictory to one another.
Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims (14)

  1. A method for generating islet β cells, comprising: coculturing endocrine progenitor cells with medium containing an M3 muscarinic acetylcholine receptor agonist, so as to facilitate the differentiation of endocrine progenitor cells into islet β cells, wherein the coculture does not occur in the differentiation process from primitive gut tube cells to endocrine progenitor cells.
  2. The method of claim 1, wherein, the endocrine progenitor cells are obtained by differentiation from the primitive gut tube cells, and in the differentiation process from the primitive gut tube cells to the endocrine progenitor cells, the cells are not cocultured with medium containing the M3 muscarinic acetylcholine receptor agonist.
  3. The method of claim 1 or 2, wherein the M3 muscarinic acetylcholine receptor agonist is acetylcholine.
  4. The method of claim 1, wherein coculturing the endocrine progenitor cells with medium containing the M3 muscarinic acetylcholine receptor agonist is accomplished as follows:
    the endocrine progenitor cells are differentiated and cultured in S6 complete medium, the S6 complete medium is MCDB131 medium supplemented with 25-200 μM acetylcholine, preferably, the S6 complete medium is MCDB131 medium supplemented with 25-100 μM acetylcholine;
    optionally, the S6 complete medium further comprises: 8 mM D- (+) -glucose, 1.23 g/L NaHCO 3 + 2%BSA, ITS-X at a dilution ratio of 1: 200, 2 mM Glutamax, 0.25 mM vitamin C, and 1%Pen/Strep.
  5. The method of claim 1, wherein the endocrine progenitor cells are obtained as follows:
    the primitive gut tube cells are cultured in S3 complete medium for 2 days to generate PDX1 positive pancreatic progenitor cells;
    the PDX1 positive pancreatic progenitor cells are cultured in S4 complete medium for 5 days to obtain PDX1/NKX6.1 double positive pancreatic progenitor cells;
    the PDX1/NKX6.1 double positive pancreatic progenitor cells are cultured in S5 complete medium for 7 days to obtain the endocrine progenitor cells.
  6. The method of claim 1, wherein the primitive gut tube cells are obtained as follows:
    the starting stem cells are inoculated and maintained in mTeSR1 medium, the mTeSR1 medium contains 10 μM Y27632, wherein the cell density of the starting stem cells in a six-well plate is 0.5*10^6 cells/well;
    the inoculated starting stem cells are cultured for 2 days at 37℃. and 5%CO 2, preferably, a half medium change method is adopted in the culture process;
    the subsequent stage 1 and 2 differentiation are performed, adopting half medium change method, so as to obtain the primitive gut tube cells, wherein the stage 1 differentiation lasts for 3 days in S1 complete medium,  and the stage 2 differentiation lasts for 3 days in S2 complete medium.
  7. The method of claim 6, wherein the starting stem cell is a commercial stem cell line, optionally, the commercial stem cell line is a H9 cell line.
  8. The method of claim 6, wherein the starting stem cells are pre-expanded;
    optionally, the expansion is performed as follows:
    H9 cell clusters are cultured in suspension in mTeSR1 medium;
    the H9 cell clusters after suspension culture are digested to obtain H9 single cells;
    the H9 single cells are inoculated into mTeSR1 medium at a density of 6*10^5 cells/mL and cultured, preferably, the half medium change method is adopted in the culture process;
    optionally, karyotype identification, flow cytometry detection and mycoplasma contamination detection are performed on the cultured cells;
    wherein, the karyotype identification result is normal, the flow cytometry result shows that the proportions of OCT4 and SSEA4 positive cells are not less than 95%, and there is no mycoplasma contamination, indicating that the cultured cells are starting stem cells for differentiation.
  9. An islet β cell obtained by the method of any one of claims 1 to 8.
  10. An islet β cell cluster, wherein, in the cell cluster, the ratio of NKX6.1/C peptide double positive is 20%～50%, preferably, the ratio of NKX6.1/C peptide double positive is 20%～40%.
  11. A pharmaceutical composition comprising the islet β cell of claim 9 or the islet β cell cluster of claim 10.
  12. Use of the islet β cell of claim 9, the islet β cell cluster of claim 10, or the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating or preventing diabetes.
  13. A method of treating or preventing diabetes in a subject comprising administering to the subject a therapeutically effective amount of the islet β cell of claim 9, the islet β cell cluster of claim 10, or the pharmaceutical composition of claim 11.
  14. The islet β cell of claim 9, the islet β cell cluster of claim 10, or the pharmaceutical composition of claim 11 for use in treating or preventing diabetes.
PCT/CN2022/127805 2021-10-28 2022-10-27 A METHOD FOR PROMOTING THE DIFFERENTIATION OF STEM CELLS INTO FUNCTIONALLY MATURE ISLET β CELLS AND USE THEREOF WO2023072170A1 (en)

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